PROJECT ABSTRACT The thirteen phospholipase C isozymes in humans hydrolyze phosphatidylinositol 4,5-bisphosphate to generate the second messengers diacylglycerol and inositol 1,4,5-trisphosphate. Hydrolysis is tightly controlled ? most PLCs are autoinhibited and activated in response to specific stimuli. The two PLC-? isozymes are uniquely activated upon phosphorylation by a diverse array of tyrosine kinases to control a wide range of biological processes including embryogenesis, immune responses, platelet aggregation, wound healing, and neuronal transmission. Conversely, constitutively active forms of the PLC-? isozymes contribute to immune dysregulation and cancer. However, despite the central importance of the PLC-? isozymes to human health, their regulation remains ill-defined and hampers efforts to treat related diseases. This application proposes a multidisciplinary approach to produce a comprehensive, mechanistic framework describing the regulated activation of the PLC-? isozymes and to use this information to control PLC-? isozymes in cells. These efforts will be greatly facilitated by the first, atomic-resolution structure of a full-length, autoinhibited PLC-? isozyme presented as preliminary data. While this structure and supporting cellular, biochemical, and biophysical studies provide important information on the regulation of the PLC-? isozymes, critical details describing the process of activation remain unknown. Most importantly, there is a paucity of information on both the intermediates that transiently populate the pathway, as well as essentially no information on the active forms of the PLC-? isozymes ultimately needed to engage membranes and hydrolyze substrate. Consequently, Aim 1 will extend the original crystallographic studies to define atomic-resolution structures of active forms of PLC-?1. In Aim 2, the autoinhibited structure of PLC-?1 will be used in conjunction with accelerated molecular dynamics simulations and hydrogen-deuterium exchange mass spectrometry to define the dynamics of the structural transitions culminating in active PLC-? isozymes. The preliminary studies have also guided the mutational dissection of PLC-?1 to allow graded control of its phospholipase activity in cells. In Aim 3, this information will be combined with state-of-the-art microfluidics and live-cell imaging to define the roles of PLC-?1 during mesenchymal chemotaxis. Ultimately, these studies will produced a detailed, comprehensive model of the regulated activation of the PLC- ? isozymes that will be used to better understand basic biological processes controlled by these phospholipases. This information is needed to treat human diseases caused by the aberrant regulation of the PLC-? isozymes.